Screening And Identification Of Antibacterial Endophytes From Single Clump Tea

The following items were carried out in this research: surface sterilization of single clump tea,isolation and purification of endophytes, screening and identification of antibacterial endophytes,determination for the antagonistic energy of ferment fitrate and optimization of fermentation conditions,The aim of this study was to obtain biological control strains which have significant inhibition activity against tested bacteria and plant pathogenic microorganism in order to provide theoretical basis for research and development of new biological antibacterial agent. The results as follows:Twenty-nine endophyte strains were isolated from Fenghuang single clump tea whose altitude is 750 m,including twenty-six endophytic bacteria FH01~FH26, one endophytic actinomycete FX15 and two endophytic fungi F15 GEN and F15 YE. Thirty-one endophytic bacteria LA1~LA31 were isolated from Lingtou single clump tea whose altitude is 750 m, Twenty-seven endophyte strains were isolated from Lingtou single clump tea whose altitude is 400 m, including twenty-five endophytic bacteria LB1~LB25and two endophytic fungi LB35 JING and LB35 JIAO.Inhibition activity experiments of endophytic bacteria against Escherichia coli, Bacillus subtilis,Staphylococcus aureus and Moniliaalbican were made by double-plate method. The results as follows: all strains didn’t had inhibition activity against Moniliaalbican. FH04 had inhibition activity against Escherichia coli. FH01, FH02, FH15, LA5 and F15 YE had inhibition activity against Bacillus subtilis.FH01, FH15, LA16 and F15 GEN had inhibition activity against Staphylococcus aureus.Inhibition activity experiments of endophytic bacteria against plant pathogenic microorganism were made by flat-stand method. The results were as follows: there were eleven endophytic bacteria had inhibition activity against Alternaria alternata, eleven endophytic bacteria had inhibition activity against Sphaerulina juglandis, twelve endophytic bacteria had inhibition activity against Alternaria sp., four endophytic bacteria had inhibition activity against Fusarium solani, ten endophytic bacteria had inhibition activity against Rhizoctonia solani, ten endophytic bacteria had weakly inhibition activity against Pythium aphanidermatum form eighty-one endophyte strains. The hyphae growth inhibition rates of all those strains,which were FH01, LA4, LA16 and LB21(against Alternaria alternata), FH01, FH04, FH17, LA3, LA4 and LB6(against Sphaerulina juglandis), LA4 and LB6(against Fusarium solani), LA3, LA4 and LB6(against Alternaria sp.), FH03, FH06, FH17 and LB6(against Fusarium solani), FH05 and LA4(against Rhizoctonia solani)were more than 80%. LB35 JING and F15 GEN had inhibition activity against Rhizoctonia solani and Valsa ambiens Pers. F15 YE had inhibition activity against Sphaerulina juglandis.LB35 JING had inhibition activity against Fusarium solani.Through morphologic observations, physiological-biochemical characters and the sequence analysis of the 16 Sr DNA: FH01 was identified as Bacillus methylotrophicus strain, FH02 was identified as Pseudomonas sp. FH04, FH15 and LA4 were identified as Bacillus sp., FH05 was identified as Serratia marcescens strain, FH17 was identified as Staphylococcus sp, LA5 was identified as Bacillus cereus, LA16 was identified as Arthrobacter sp, LB21 was identified as Serratia sp. Through morphologic observations and the sequence analysis of the 18 SrDNA: F15 GEN was identified as Penicillium decumbens, F15 YE and LB35 JING were identified as Penicillium sp. respectively.The preliminary determination showed that the antibacterial biological titer for ferment fitrate of FH01,FH02, FH15 and LA5 to Bacillus subtilis were 5.33, 3.42, 2.19 and 2.47μg/mg; FH01, FH15 and LA16 to Staphylococcus aureus were 10.91, 6.49 and 20.71μg/mg; FH03, FH06, FH17, LA3 and LB6 to Fusarium solani were 866, 931, 917, 579 and 870U/m L; FH01, FH02, FH04, FH05, FH15, FH17, LA3, LA4, LA16 and LA19 to Rhizoctonia solani were 715, 295, 2512, 10806, 3282, 94, 1354, 8518, 4988 and 1489U/mL.The optimum fermentation conditions of FH01 showed that the adding amount of malt extract was1.43%(mg/m L), the adding amount of soy peptone was 1.67%(mg/m L), K2HP04·3H20 was 0.3%(mg/m L),the p H was 5.0, the fermentation temperature was 36.78 ℃, the shaking speed was 129.52 rpm, the fermentation time was 67.36 h. In this condition, the diameters of inhibitory zones was 22.10 mm, which was 34.39% higher than the initial conditions(14.50 mm) and compared with the predicted value of 24.10 mm, the difference is only 2 mm.